The cell death machinery is conserved throughout evolution and is composed of activators, inhibitors, and effectors (Chinnaiyan, A. M. and Dixit, V. M., Curr. Biol. 6:555–562 (1996)). The effector arm of the cell death pathway is composed of a rapidly growing family of cysteine aspartate-specific proteases termed caspases (Alnemri, E. S., et al., Cell 87:171 (1996)). As implied by the name, these cysteine proteases cleave substrates following an aspartate residue (Alnemri, E. S., et al., Cell 87:171 (1996); Walker, N. P., et al., Cell 78:343–352 (1994)). Caspases are normally present as single polypeptide zymogens and contain an amino-terminal prodomain, and large and small catalytic subunits (Wilson, K. P., et al., Nature 370:270–274 (1994); Rotonda, J., et al., Nat. Struct. Biol. 3:619–625 (1996); Fraser, A. and Evan, G., Cell 85:781–784 (1996)). The two chain active enzyme (composed of the large and small subunits) is obtained following proteolytic processing at internal Asp residues (Wilson, K. P., et al., Nature 370:270–274 (1994); Rotonda, J., et al., Nat. Struct. Biol. 3:619–625 (1996); Fraser, A. and Evan, G., Cell 85:781–784 (1996)). As such, caspases are capable of activating each other in a manner analogous to zymogen activation that is observed in the coagulation cascade (Boldin, M. P., et al., Cell 85:805–815 (1996)). The identification of FLICE and Mch4/FLICE2 as receptor associated caspases suggested a surprisingly direct mechanism for activation of the death pathway by the cytotoxic receptors CD-95 and TNFR-1 (Boldin, M. P., et al., Cell 85:805–815 (1996); Muzio, M., et al., Cell 85:817–827 (1996); Vincenz, C. and Dixit, V. M., J. Biol. Chem. 272:6578–6583 (1997); Chinnaiyan, A. M., et al., Cell 81:505–512 (1995)). Upon activation, both receptors use their death domains to bind the corresponding domain in the adaptor molecule FADD (Fas-associated death domain protein) (Muzio, M., et al., Cell 85:817–827 (1996); Vincenz, C. and Dixit, V. M., J. Biol. Chem. 272:6578–6583 (1997); Chinnaiyan, A. M., et al., Cell 81:505–512 (1995)). Dominant negative versions of FADD that lack the N-terminal segment but still retain the death domain potently inhibit both CD-95 and TNFR-1 induced apoptosis (Chinnaiyan, A. M., et al., J. Biol. Chem. 271:4961–4965 (1996); Muzio, M., et al., J. Biol. Chem. 272:2952–2956 (1997)). Given the importance of the N-terminal segment in engaging the death pathway, it has been termed the death effector domain (DED) (Chinnaiyan, A. M., et al., J. Biol. Chem. 271:4961–4965 (1996)).
Remarkably, the DED is present within the prodomain of FLICE and Mch4/FLICE2 and mutagenesis studies suggest that a homophilic interaction between the DED of FADD and the corresponding domain in FLICE or Mch4/FLICE2 is responsible for the recruitment of these proteases to the CD-95 and TNFR-1 signaling complexes (Muzio, M., et al., Cell 85:817–827 (1996); Vincenz, C. and Dixit, V. M., J. Biol. Chem. 272:6578–6583 (1997); Chinnaiyan, A. M., et al., Cell 81:505–512 (1995); Chinnaiyan, A. M., et al., J. Biol. Chem. 271:4961–4965 (1996)). Taken together, these data are consistent with FLICE and Mch4/FLICE2 being apical enzymes that initiate precipitous proteolytic processing of downstream caspases resulting in apoptosis (Boldin, M. P., et al., Cell 85:805–815 (1996); Srinivasula, S. M., et al., PNAS 93:14486–14491 (1996); Fernandes-Alnemri, T., et al., PNAS 93:7464–7469 (1996); Henkart, P. A., Immunity 4:195–201 (1996)). A number of viral gene products antagonize CD-95 and TNFR-1 mediated killing as a means to persist in the infected host (Shen, Y. and Shenk, T. S., Current Opinion in Genetics and Development 5:105–111 (1995)). The poxvirus encoded serpin CrmA and baculovirus gene product p35 are direct caspase inhibitors (Walker, N. P., et al., Cell 78:343–352 (1994)). In contrast, the molluscum contagiosum virus protein MC159 and the equine herpes virus protein E8 encode DED-containing decoy molecules that bind to either FADD (MC159) or FLICE (E8) and disrupt assembly of the receptor signaling complex, thereby abrogating the death signal (Hu, S., et al., J. Biol. Chem. 272:9621–9624 (1997); Bertin, J., et al., PNAS 94:1172–1176 (1997); Thome, M., et al., Nature 386:527–521 (1997)). The existence of these viral inhibitors has raised the question of whether functionally equivalent molecules are encoded in the mammalian genome.
There is a need for factors, such as the polypeptides of the present invention, that are useful for inhibiting apoptosis for therapeutic purposes, for example, in the treatment of Alzheimer's disease, Parkinson's disease, rheumatoid arthritis, septic shock, sepsis, stroke, CNS inflammation, osteoporosis, ischemia, reperfusion injury, cell death associated with cardiovascular disease, polycystic kidney disease, apoptosis of endothelial cells in cardiovascular disease, degenerative liver disease, MS and head injury damage. There is a need, therefore, for the identification and characterization of such factors that are inhibitors of apoptosis, such as the I-FLICE-1 and I-FLICE-2 polypeptides of the present invention, which can play a role in preventing, ameliorating or correcting the diseases and disorders associated with apoptosis.